Interferon-γ modulates lung macrophage production of PDGF-BB and fibroblast growth

Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. We have studied here the cytokine interferon-γ (IFN-γ) which is known to prime macrophages for increased PDGF production. Thus, we postulated that IFN-γ would act as a positive regulator of PDGF-BB secretion by rat alveolar macrophages, and in addition we asked whether or not the IFN-γ (a known anti-mitogenic cytokine) would block the growth response of primary lung fibroblasts to the PDGF-BB. Macrophages incubated with IFN-γ or iron spheres alone for 24 h secreted 2.5-fold more PDGF-BB than control macrophages incubated in serum-free medium. Preincubation of macrophages with IFN-γ prior to the addition of iron spheres synergistically increased PDGF-BB production 2–10-fold after 24 h. In contrast, when IFN-γ was added to quiescent rat lung fibroblasts (RLFs) in the presence of PDGF-BB, the cytokine induced a concentration-dependent decrease in cell growth, while IFN-γ alone did not affect proliferation. [¹²⁵I]PDGF-BB receptor assays showed that neither preincubation nor coincubation of RLF with IFN-γ affected PDGF-BB binding to its receptors.     

Effects of transforming growth factor β1 and basic fibroblast growth factor on lipoprotein lipase activity in primary cultures of chicken (Gallus domesticus) adipocyte precursors

• 1.1. The effect of TGF-β and bFGF on lipoprotein lipase activity in chicken adipocyte precursors was investigated.
• 2.2. Lipoprotein lipase activity was reduced by up to 80% by incubation with TGF-β whereas bFGF had no effect.
• 3.3. Contrary to that found with the 3T3-L1 preadipocyte cell line it was not necessary for TGF-β to be present prior to the start of differentiation in order to be effective.
• 4.4. Incubation of adipocyte precursors with actinomycin D abolished the effect of TGF-β suggesting that synthesis of a protein effector is required.
• 5.5. These results indicate differences in responsiveness to TGF-β and bFGF between primary chicken adipocyte precursors and some preadipocyte cell lines.

     

Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in normal human seminiferous epithelium

 Fibroblast growth factor 2 (FGF-2), which occurs in various isoforms both species and tissue specifically, regulates cell proliferation and differentiation via a dual receptor system consisting of heparan sulphate proteoglycans and receptor tyrosine kinases (FGFRs). This study demonstrates for the first time the distribution pattern of FGF-2 and the receptors FGFR 1–4 in the normal seminiferous epithelium of adult men. In western blot analyses, the polyclonal antibody, anti-FGF-2, shows two immunoreactive bands at 18 and 24 kDa. On paraffin sections, positive immunoreaction occurs within the cytoplasm of spermatogonia. The distribution pattern of the polyclonal anti-FGFR 1–4 antibodies is as follows: anti-FGFR-1 (one 68-kDa band) stains nuclei and cytoplasm of spermatogonia; anti-FGFR-3 (five bands at 68, 78, 105, 125 and 145 kDa) stains the nuclei of all germ cells except those of elongated spermatids; and anti-FGFR-4 (one 48-kDa band) stains the cytoplasm of primary pachytene spermatocytes. We were unable to demonstrate FGFR-2 immunoreactivity either in western blot analysis or on paraffin sections. This distribution pattern suggests that FGF-2 in spermatogonia is involved in the autocrine and paracrine regulation of the proliferation and differentiation of spermatogonia and spermatocytes via the receptors FGFR-1, FGFR-3 and FGFR-4. Accepted: 23 December 1997     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Calbindin 1, fibroblast growth factor 20, and α-synuclein in sporadic Parkinson’s disease

Parkinson’s disease (PD), one of the most common human neurodegenerative disorders, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Our recent case-control association study of 268 SNPs in 121 candidate genes identified α-synuclein (SNCA) as a susceptibility gene for sporadic PD (P = 1.7 × 10⁻¹¹). We also replicated the association of fibroblast growth factor 20 (FGF20) with PD (P = 0.0089). To find other susceptibility genes, we added 34 SNPs to the previous screen. Of 302 SNPs in a total 137 genes, but excluding SNCA, SNPs in NDUFV2, FGF2, CALB1 and B2M showed significant association (P < 0.01; 882 cases and 938 control subjects). We replicated the association analysis for these SNPs in a second independent sample set (521 cases and 1,003 control subjects). One SNP, rs1805874 in calbindin 1 (CALB1), showed significance in both analyses (P = 7.1 × 10⁻⁵; recessive model). When the analysis was stratified relative to the SNCA genotype, the odds ratio of CALB1 tended to increase according to the number of protective alleles in SNCA. In contrast, FGF20 was significant only in the subgroup of SNCA homozygote of risk allele. CALB1 is a calcium-binding protein that widely is expressed in neurons. A relative sparing of CALB1-positive dopaminergic neurons is observed in PD brains, compared with CALB1-negative neurons. Our genetic analysis suggests that CALB1 is associated with PD independently of SNCA, and that FGF20 is associated with PD synergistically with SNCA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synergistic effects of endothelin-1 (ET-1) and transforming growth factor alpha (TGF-α) or epidermal growth factor (EGF) on DNA replication and G1 to S phase transition

The cooperative cell kinetic actions of ET-1 with TGF- or EGF in normal rat kidney fibroblasts (NRK-49F) and KNRK cells (Kirsten MSV transformed) were analyzed by [³H]-thymidine incorporation assay and flow cytometry. A marked synergistic effect of TGF- and ET-1 (or EGF and ET-1) on DNA synthesis and G1 to S transition was observed in NRK cells; 15–20% S for TGF- and 12% S for ET-1 alone but 45–50% S in combination. There was no detectable effect on cell cycle kinetics by TGF- (1 ng/ml) or EGF (1 ng/ml) plus ET-1 (1 ng/ml) in KNRK cells treated for 22 hours. Insulin, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and transforming growth factor (TGF-) were also tested and found to have no significant synergistic effects on ET-1 actions. Our findings suggest that the combination of TGF- (EGF) and ET-1 is an important part of an intricate network which coordinates progression of G1 to S phase in normal cells.     

Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism?

Basic fibroblast growth factor, (bFGF), promotes the formation of new blood capillaries and is sequestered and protected by binding to heparan sulfate (HS), both on the cell surface and in the extracellular matrix. Release of HS-bound bFGF by heparin-like molecules and HS-degrading enzymes (i.e., heparanase) provides a novel mechanism for regulation of the growth of capillary blood vessels in normal and pathological situations. The extracellular matrix also serves as a storage depot for other growth factors and enzymes.     

Molecular cloning and developmental expression of rat fibroblast growth factor receptor 3

Fibroblast growth factors (FGFs) are involved in the control of a variety of biological functions including regulation and differentiation of various cell types. Furthermore, they play important roles in the processes of regeneration, angiogenesis, and chemotaxis. The family of FGF receptors (FGFRs) comprises four members, FGFR-1 to -4, which exist in several differentially expressed splice variants. Except for FGFR-3, primary structures and expression of the three other FGFRs have been described in the rat system. Although expression studies with heterologous probes of FGFR-3 from mice have been performed in the rat system, these analyses were limited and the complete set of receptors has not yet been revealed. To understand the developmental functions of FGFR-3, it is important to elucidate the expression pattern in embryos of different stages. In this study, we have isolated a cDNA of FGFR-3 from rat brain. Expression analyses by RT-PCR of adult rat revealed expression in several tissues, however, expression levels were highest in lung and brain. During embryonic development, FGFR-3 displays a diffuse expression in most tissues at embryonic day 14 (E14), as observed by in situ hybridization experiments. In E18 the expression pattern is more restricted, showing strong signals in spinal cord, dorsal root ganglia, cortex, chondrocytes, and endothelial cells. The temporal and spatial pattern of FGFR-3 expression suggests specific functions in several tissues during development.     

Basic fibroblast growth factor is a β-rich protein

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The far-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel β-sheets. Analysis of the CD spectrum by the least-squares method of Changet al. (1978) and the CONTIN program of Provencher and Glöckner (1981) suggested that about one half of the molecule consisted of β-sheet and there was no α-helix. These estimates agreed with the prediction by the sequence method of Garnieret al. (1978) using decision constants based on CD results. bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains atpH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type.     

Transforming growth factor-β1 (TGF-β1) and acetylcholine (ACh) alter nitric oxide (NO) and interleukin-1β (IL-1β) secretion in human colon adenocarcinoma cells

Colon adenocarcinoma is one of the most common fatal malignancies in Western countries. Progression of this cancer is dependent on tumor microenvironmental signaling molecules such as transforming growth factor-β (TGF-β) or acetylcholine (ACh). The present study was conducted to assess the influence of recombinant human transforming growth factor (rhTGF)-β1 or ACh on nitric oxide (NO) and interleukin-1β (IL-1β) secretion by three human colon adenocarcinoma cell lines: HT29, LS180, and SW948, derived from different grade tumors (Duke’s stage). The cells were cultured in 2D and 3D (spheroids) conditions. Colon carcinoma cells exhibited different sensitivities to rhTGF-β1 or ACh dependent on the tumor grade and the culture model. ACh exhibited significant inhibitory effects towards NO, endothelial nitric oxide synthase (eNOS), and IL-1β secretion especially by tumor cells derived form Duke’s C stage of colon carcinoma. rhTGF-β1 also decreased NO, IL-1β, and eNOS expression, but its effect was lower than that observed after the administration of ACh. The inhibition of NO and IL-1β production was more striking in 3D tumor spheroids than in 2D culture monolayers. Taken together, the TGF-β1–ACh axis may regulate colon carcinoma progression and metastasis by altering NO secretion and influence inflammatory responses by modulating IL-1β production.     

Enhanced fibroblast contraction of 3D collagen lattices and integrin expression by TGF-beta1 and -beta3: mechanoregulatory growth factors?

Generation of contractile forces as fibroblasts attach and migrate through collagenous substrates is a fundamental behavior, yet its regulation and consequences are obscure. Although the transforming growth factor-betas (TGF-beta) are similarly important in fibrosis and tissue repair, their role in contraction is controversial. Using a quantitative, 3D collagen culture model we have measured the effects of TGF-beta1 and -beta3 on contractile forces generated by human dermal fibroblasts. Maximal stimulation was between 7.5 and 15 ng/ml of TGF-beta1. Higher doses were inhibitory (30 ng/ml), giving a bell-shaped dose response. The initial rate of force generation was increased sevenfold (15 ng/ml). A similar response pattern was seen with TGF-beta3 alone. However, the addition of both isoforms together stimulated a biphasic increase in force generation, suggesting that there was a distinct temporal cooperativity between the two isforms. This very early onset (10-20 min) of stimulation suggested that TGF-beta might act through cell attachment and integrin function and the effect of TFG-beta on expression of fibronectin (FnR) and vitronectin (VnR) integrin receptors was monitored over the same time scale. TGF-beta1 dramatically up-regulated VnR expression, relative to FnR, over time but the optimal time for this was 2-4 h later than that of force stimulation. It is concluded that TGF-beta1 and -beta3 behave here primarily as mechanoregulatory growth factors and that stimulation of integrin expression may be a consequence of the altered cell stress.     

Fibronectin induces endothelial cell migration through β1 integrin and Src-dependent phosphorylation of fibroblast growth factor receptor-1 at tyrosines 653/654 and 766

The extracellular matrix microenvironment regulates cell phenotype and function. One mechanism by which this is achieved is the transactivation of receptor tyrosine kinases by specific matrix molecules. Here, we demonstrate that the provisional matrix protein, fibronectin (FN), activates fibroblast growth factor (FGF) receptor-1 (FGFR1) independent of FGF ligand in liver endothelial cells. FN activation of FGFR1 requires β1 integrin, as evidenced by neutralizing antibody and siRNA-based studies. Complementary genetic and pharmacologic approaches identify that the non-receptor tyrosine kinase Src is required for FN transactivation of FGFR1. Whereas FGF ligand-induced phosphorylation of FGFR1 preferentially activates ERK, FN-induced phosphorylation of FGFR1 preferentially activates AKT, indicating differential downstream signaling of FGFR1 in response to alternate stimuli. Mutation analysis of known tyrosine residues of FGFR1 reveals that tyrosine 653/654 and 766 residues are required for FN-FGFR1 activation of AKT and chemotaxis. Thus, our study mechanistically dissects a new signaling pathway by which FN achieves endothelial cell chemotaxis, demonstrates how differential phosphorylation profiles of FGFR1 can achieve alternate downstream signals, and, more broadly, highlights the diversity of mechanisms by which the extracellular matrix microenvironment regulates cell behavior through transactivation of receptor tyrosine kinases.     

Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: implications for human efficacy and toxicity

Glucosamine is used for alleviating pain in osteoarthritis. Clinical trials have reported that glucosamine has equivocal efficacy. Glucosamine is also used in cell cultures to stimulate hexosamine flux and protein O-glycosylation, but at many-fold greater concentrations than those in human plasma following oral dosing. Lean Zucker rats were dosed orally for 6 weeks with glucosamine hydrochloride at doses (0–600 mg/kg/day) that produced peak serum concentrations of <1–35 μM, spanning the human exposure range. Relative expression of both TGFβ1 and CTGF mRNA were significantly increased up to 2.3-fold in liver, kidney and articular cartilage when evaluated 4 h after final dose. Apparent threshold serum glucosamine (C_max) concentration required to increase TGFβ1 expression in cartilage was 10–20 μM. These increases were associated with significant increases in UDP-N-acetylglucosamine concentrations suggesting increased hexosamine flux. Both TGFβ1 and CTGF are mediators of chondrocyte proliferation and cartilage repair. Study demonstrates that oral glucosamine doses that produce clinically relevant serum glucosamine concentrations can induce tissue TGFβ1 and CTGF expression in vivo and provides a mechanistic rationale for reported beneficial effects of glucosamine therapy. Induction of renal TGFβ1 and CTGF mRNA suggests that potential sclerotic side-effects may occur following consumption of potent glucosamine preparations.     

Is serum fibroblast growth factor 21 associated with the severity or presence of coronary artery disease?

BackgroundRecent studies have shown that increased circulating concentrations of fibroblast growth factor 21 (FGF21) are associated with obesity, metabolic disorder, and atherosclerosis. However the relationship between FGF21 and coronary artery disease (CAD) is controversial This study was planned to investigate the role of FGF21 in CAD development and CAD severity.MethodsSeventy-eight patients with stable angina pectoris (SAP) (lesion positive) and 40 control patients (lesion negative) with similar cardiovascular risk factors were included in the study. Serum FGF21 levels were measured by ELISA method. CAD severity was evaluated by using SYNTAX and GENSINI risk scores.ResultsFGF21 concentrations were found significantly higher in the SAP group than in the control group. [101.18 ± 141.62 vs. 47.93 ± 58.74 pg/mL; p = 0.03], no correlation was found between the SYNTAX (r = 0.146 and p = 0.134) and GENSINI (r = 0.211 and p = 0.084) scores with serum FGF21 levels. There was a negative relationship between serum FGF21 and serum HDL-C levels in correlation analysis (r = - 0.272; p = 0.026).ConclusionsThe serum FGF21 levels are different between SAP and control patients. FGF21 is a marker for CAD diagnosis, but not for the evaluation of CAD severity.     

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-β1 (TGF-β1) administration suppresses osteoblast differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.     

Antiproliferative heparan sulfate inhibiting hyaluronan and transforming growth factor-β expression in human lung fibroblast cells

The objective of this study was to examine the effects of heparan sulfate (HS) on factors involved in the remodeling of connective tissue observed in patients with fibrotic respiratory disorders such as asthma. A suitable working model is to stimulate human fetal lung fibroblasts in vitro with structurally different forms of HS. Highly sulfated and iduronic acid (IdoUA)-rich HS specifically decreased cell proliferaton, production of jyaluronan (HA), transforming growth factor (TGF)-β₁, and TFF-β-induced α-smooth muscle actin but did not affect the overall proteoglycan production in the cells. These repressed factors are suggested to play a critical role in the early stages of remodeling and myofibroblast activation. Low sulfated and IdoUA-poor HS did not display any effects on these factors. Furthermore, analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a 70% increased expression of annexin II, which has previously been shown to have a high affinity for both heparin and HS. Heat-shock protein 27 and arsenite translocating factor, both involved in actin organization and polymerization, were also increased in the HS-stimulated cells. Thus, the reduced expression of HA and TGF-β₁, both important in the development of fibrosis, seems to be mediated by pecific changes in protein expression of the fibroblast. The observed inhibition of cell proliferation, HA, and TGF-β₁ allows speculation of highly sulfated HS as a antifibrotic candidate in the early stage of remodeling.     

Effects of concomitant use of fibroblast growth factor (FGF)-2 with beta-tricalcium phosphate (β-TCP) on the beagle dog 1-wall periodontal defect model

The effects of concomitant use of fibroblast growth factor-2 (FGF-2) and beta-tricalcium phosphate (β-TCP) on periodontal regeneration were investigated in the beagle dog 1-wall periodontal defect model. One-wall periodontal defects were created in the mesial portion of both sides of the mandibular first molars, and 0.3% FGF-2 plus β-TCP or β-TCP alone was administered. Radiographic evaluation was performed at 0, 3, and 6 weeks. At 6 weeks, the periodontium with the defect site was removed and histologically analyzed. Radiographic findings showed that co-administration of FGF-2 significantly increased bone mineral contents of the defect sites compared with β-TCP alone. Histologic analysis revealed that the length of the regenerated periodontal ligament, the cementum, distance to the junctional epithelium, new bone height, and area of newly formed bone were significantly increased in the FGF-2 group. No abnormal inflammatory response or ankylosis was observed in either group. These findings indicate the efficacy of concomitant use of FGF-2 and β-TCP as an osteoconductive material for periodontal regeneration following severe destruction by progressive periodontitis.